1. Field of the Invention
This invention relates to a hybridoma cell line and monoclonal antibody produced thereby which may be used to detect eggs of the corn earworm, Helicoverpa zea.
2. Description of the Prior Art
Both the corn earworm (also known as the cotton bollworm), Helicoverpa zea, and the tobacco budworm, Heliothis virescens, infest cotton fields in Texas, midsouth United States, and Mexico. If uncontrolled, these two species, commonly referred to as the budworm/bollworm complex, have the potential to inflict very substantial yield losses at harvest. However, efforts to control the insects have been hampered by the tendency of the budworm, H. virescens, to become resistant to pyrethroid insecticides. As yet, the corn earworm, H. zea, has not exhibited resistance to pyrethroids.
Pyrethroid resistance in H. virescens in cotton was first detected in California in the early 1980s and in the midsouth and in Texas in the mid 1980s (Gage et al., 1991, Intro. Southwest. Entomol. Suppl., 15:1-3), and has increased in intensity in several regions during subsequent years (Carillo, 1991, Proceedings, Beltwide Cotton Production Res. Conference, Nashville, Tenn., Jan. 2-7, 1989, National Cotton Council of America, Nashville, Tenn., pp. 59-67; Elzen et al., 1991, Southwest. Entomol. Suppl., 15:27-31; and Graves et al., 1991, Southwest. Entomol. Suppl., 15:33-41).
Levels of pyrethroid resistance vary temporally and geographically, tending to begin low and increase during the growing season, and to be higher in cotton growing than in noncotton growing areas (Graves ibid; Lettrell et al., 1991, Southwest. Entomol. Suppl., 15:5-26; and Plapp, 1991, Southwest. Entomol. Suppl., 15:69-73). These trends indicate not only that the evolution of resistance is promoted by pyrethroid use, but that when selection pressure is relaxed, genes for pyrethroid resistance confer reduced fitness. This interpretation is supported by laboratory studies demonstrating reduced female attractiveness, reduced fecundity, and increased development times in resistant populations (Campanhola and Plapp, 1989, Proceedings, Beltwide Cotton Production Res. Conference, Nashville, Tenn., Jan. 2-7, 1989, National Cotton Council of America, Nashville, Tenn., pp. 352-359; and McCutcheon et al., 1989, Proceedings, Beltwide Cotton Production Res. Conference, Nashville, Tenn., Jan. 2-7, 1989, National Cotton Council of America, Nashville, Tenn., pp. 364-366). Hence a reduction of pyrethroid use can be expected to retard the development of resistance and extend the useful life of these chemicals (Mallett and Luttrell, 1991, Southwest. Entomol. Suppl., 15:201-212).
Because cotton fields may be infested with either or both of H. zea or H. virescens, control efforts, particularly the selection of an appropriate pesticide and the amount applied, would be aided by the ability to accurately differentiate between these two closely related pests at an early stage, preferably as eggs. However, there are currently no means available for readily differentiating between eggs of H. zea and H. virescens in the field. This has often resulted in the wasteful spraying of pyrethroids as well as the overexposure of populations of H. virescens to the insecticides, thereby increasing selection pressure for pyrethroid resistance. Unnecessary spraying also depresses natural enemy populations, further hindering control of infestations. Morphological differences between eggs of H. zea and H. virescens were described by Bernhardt and Phillips (1985, Southwest. Entomol., 10:236-238). However, these differences have proved too subtle to be practical for wide scale use in field determinations.
Immunoassays employing monoclonal antibodies have been developed as tools in ecological studies involving predator-prey relationships. This approach has been used to evaluate predators as potential biocontrol agents. In these studies, digestive tract contents of potential predators are subjected to immunochemical analysis to determine if they have ingested specific pest insects. One such monoclonal antibody binding the arylphorin of H. zea, which was produced to whole plasma of the fifth instar larvae of H. zea, was described by Lenz and Greenstone (1988, Arch. Insect Biochem. Physiol., 9:167-178) and Greenstone and Morgan (1989, Ann. Entomol. Soc. Am., 82:45-49). Hagler (1994, Ann. Entomol. Soc. Am., 87:85-90) described a monoclonal antibody specific for an egg antigen of the pink bollworm, Pectinophora gossypiella (Saunders). Most recently, Greenstone and Trowell (1994, Ann. Entomol. Soc. Am., 87:214-217) described two additional monoclonal antibodies produced using the purified vitellins of H. virescens and H. zea, respectively, as immunogens. Vitellins have been previously described as the major yolk proteins of insect eggs, including Lepidopterans such as H. zea and H. virescens by Hagedorn and Kunkel (1979, Ann. Rev. Entomol., 24:475-505), Harnish and White (1982, J. Exp. Zool., 220:1-10), Kunkel and Nordin [1985, Yolk Proteins, In: Comprehensive Insect Physiology, Kerkut and Gilbert (eds.), Pergamon Press, Oxford, vol. 1, pp. 83-111], Kanost et al. (1990, Adv. Insect Physiol., 22:299-396), and Raikhel and Dhadialla (1992, Ann. Rev. Entomol., 37:217-251). However, while the monoclonal antibodies of Greenstone and Trowell were prepared against the vitellins of H. zea and H. virescens, they recognize the eggs of heliothine noctuid insects in general, and they cannot differentiate between the species, nor can they differentiate between the eggs of H. zea or H. virescens.